Project Summary: During cell differentiation, transcriptional programs are changed, and then must be maintained in turn. Chromatin-based epigenetic mechanisms are at the core of maintenance and switching of transcriptional programs. The fundamental issues of the nature of epigenetic marking and of the mechanisms that switch this marking during differentiation remain unclear due to lack of relevant experimental approaches. We developed new experimental paradigms that allow investigating the structure of chromatin during DNA replication at a single-cell and at a gene-specific levels. Using our new techniques, we found striking differences in the structure of chromatin during differentiation of the pluripotent human embryonic stem cells (hESC) and the antigen-inexperienced (nave) T cells. During the first several hours after induction of differentiation of hESCs to dopamine neuron lineage, or T cells to different T cell subsets, accumulation of H3K27me3 is significantly delayed on nascent DNA. Since the occurrence of H3K27me3 in the genome coincides with the dense structure of nucleosomes, this suggests the existence of a temporarily de-condensed structure of nucleosomes on nascent DNA shortly after induction of cell differentiation. Our preliminary data indicate that the de-condensed, `open' structure of chromatin may be essential for recruitment to DNA of the lineage-specific transcription factors (TFs) that are essential to induce changes in transcriptional programs during cell differentiation. Thus, our results present a molecular explanation of how the vast areas of the repressed genome can be activated during cell differentiation. The goals of this proposal are to test two unique hypotheses using different models of differentiation to various lineages for pluripotent hESCs and for specialized T cells: 1) To examine whether the period of `open' post-replicative chromatin in early differentiating cells is a result of complex interplay of activities of several histone-modifying proteins; and 2) To examine whether this open post-replicative chromatin creates a `window of opportunity' for high accessibility of lineage- specifying TFs that are required to change the transcriptional program during cell differentiation. Examining these unique hypotheses may provide a universal chromatin-based molecular mechanism for biological plasticity of the cell.